The genus Alouatta (howler monkeys) is the most widely distributed of New World primates, and has been arranged in three species groups: the Central American Alouatta palliata group and the South American Alouatta seniculus and Alouatta caraya groups. While the latter is monotypic, the A. seniculus group encompasses at least three species (A. seniculus, A. belzebul and A. fusca). In the present study, approximately 600 base pairs of the g1-globin pseudogene were sequenced in the four Brazilian species (A. seniculus, A. belzebul, A. fusca and A. caraya). Maximum parsimony and maximum likelihood methods yielded phylogenetic trees with the same arrangement: {A. caraya [A. seniculus (A. fusca, A. belzebul)]}. The most parsimonious tree had bootstrap values greater than 82% for all groupings, and strength of grouping values of at least 2, supporting the sister clade of A. fusca and A. belzebul. The study also confirmed the presence of a 150-base pair Alu insertion element and a 1.8-kb deletion in the g1-globin pseudogene in A. fusca, features found previously in the remaining three species. The cladistic classification based on molecular data agrees with those of morphological studies, with the monospecific A. caraya group being clearly differentiated from the A. seniculus group.

INTRODUCTION

The systematics of the New World monkeys (infraorder Platyrrhini) has undergone considerable revision during the past two decades, but remains controversial (e.g. Rosenberger, 1984; Ford, 1986; Kay, 1990). Nevertheless, recent studies of DNA sequences (Schneider et al., 1993, 1996; Harada et al., 1995; Goodman, 1996; Barroso et al., 1997) have consistently supported an arrangement which recognizes only 15 genera, distributed in three families: Atelidae, Pitheciidae and Cebidae. Following this arrangement, the Atelidae encompasses four genera: Alouatta (howler monkeys), Ateles (spider monkeys), Brachyteles (muriquis) and Lagothrix (woolly monkeys). Relationships within the ateline clade - Ateles, Brachyteles and Lagothrix - have been disputed, but there is a growing consensus on the grouping of Brachyteles and Lagothrix, including conclusive evidence from gamma(g)-globin gene sequences (Meireles et al., 1999). With one exception (Kay, 1990), there is also a general consensus on the distinct position of Alouatta, which is placed separately in either its own tribe, Alouattini (Rosenberger, 1984; Ford, 1986), or subfamily, Alouattinae (Hill, 1962; Napier and Napier, 1967; Hershkovitz, 1977).

Alouatta is the most widely distributed Neotropical primate genus (Neville et al., 1988), ranging from southern Mexico to northern Argentina, and is found in tropical and subtropical forest ecosystems throughout Brazil (Hirsch et al., 1991). Hershkovitz (1949) recognized three species groups based on the structure of the hyoid apparatus: the Central American Alouatta palliata group, and the South American Alouatta seniculus and Alouatta caraya groups. While the latter group is monotypic, the other two encompass a variety of different forms, whose arrangement has been subject to conflicting interpretations (e.g. Hershkovitz, 1949; Hill, 1962; Mittermeier et al., 1988; Groves, 1993; Bonvicino et al., 1995; Stanyon et al., 1995; Rylands and Brandon-Jones, 1998).

Despite the need for further work, especially on the integration of genetic and morphological data, the most recent major revisions of the genus (Hershkovitz, 1949; Hill, 1962) all divide the A. seniculus group into three species: A. seniculus, A. belzebul (endemic to Brazil) and A. (guariba) fusca. Groves (1993) does recognize a fourth species, Alouatta sara, but it is endemic to Bolivia, and thus not relevant to the present study. More recent studies (Bonvicino et al., 1995) also allocate species status to a number of subspecific forms of both A. seniculus and A. belzebul, but in the absence of a consensus, the arrangement of Groves (1993) will be followed here.

In addition to other unresolved questions, the karyotype of Alouatta varies from 2n = 43 to 2n = 54 (Armada et al., 1987; Lima and Seuánez, 1991; Stanyon et al., 1995). Of the four Brazilian species, only A. caraya has a fixed number of chromosomes, with 2n = 52 (Pargament et al., 1984), whereas in A. seniculus, 2n varies from 43 to 49 (Yunis et al., 1976; Lima and Seuánez, 1991). A. belzebul and A. fusca, on the other hand, share both the same diploid number (2n = 49/50) and a translocation on the Y chromosome (Koiffmann and Saldanha, 1974; Armada et al., 1987).

In humans, the g-globin gene is located on the short arm of chromosome 11 (Lebo et al., 1979; Bunn and Forget, 1986). This gene is expressed embryonically in strepsirrhine primates (Tagle et al., 1988), but was duplicated in tandem in anthropoids (g1 and g2), becoming expressed fetally (Fitch et al., 1991). This globin gene is part of the beta(b)-globin gene cluster, which in primates includes the e, g, yh, d and b globin genes (Figure 1). All genes in this cluster have three exons and two introns (Lawn et al., 1980). In the atelids, g1 is a pseudogene (Meireles et al., 1995, 1998), whereas in the platyrrhine genera Aotus and Saimiri, the two loci are combined into a hybrid gene (Chiu et al., 1996).

Figure 1 - Model of the b-cluster showing the five globin genes. The g1-globin pseudogene is amplified exhibiting the primers R1 and R2 used in this study and also the 1.8-kb deletion shared by all four Alouatta species as well as by the ateline group (Meireles et al., 1995, 1998).

Several recent studies (Schneider et al., 1993, 1996; Harada et al., 1995; Porter et al., 1995, 1997a,b; Meireles et al., 1999) have used DNA sequences of the globin gene to resolve phylogenetic relationships in the platyrrhines, producing results generally consistent with those of more traditional morphological studies in most (see Schneider and Rosenberger, 1996), but not all (Shoshani et al., 1996) cases. In the present study, sequences of the g1-globin pseudogene were used in a molecular analysis of the phylogenetic relationships among Brazilian howler monkeys. The aligned sequences were also used to estimate divergence times based on a local molecular clock, and to investigate the presence of the Alu insertion - found in other Alouatta species (Meireles et al., 1995, 1998) - in A. fusca.

MATERIAL AND METHODS

Study species

Howler monkeys are large-bodied, prehensile-tailed platyrrhines more specialized for folivory than frugivory, as is typical of other atelids. They are also highly sexually dimorphic in body size (adult males are 70-80% heavier than females), and two of the Brazilian species (A. caraya and A. fusca) are among only three platyrrhines (the other is Pithecia pithecia) to exhibit sexual dimorphism in pelage coloration (Mittermeier et al., 1988). A. belzebul also exhibits the most marked individual variation in pelage coloration of any platyrrhine (Bonvicino et al., 1989). Only one species (A. belzebul) is endemic to Brazil, but it is also the only one found in both Amazonian and Atlantic forest biomes. The remaining three species are found in Amazonia (A. seniculus), the Brazilian Atlantic forest and neighboring areas (A. fusca), and the savanna and swampy habitats of central and western Brazil (A. caraya).

Preparation and amplification of DNA

Genomic DNA was extracted from the peripheral blood cells of captive specimens of the four Brazilian howler monkey species (Table I), and the g1-globin sequences for A. caraya, A. belzebul and A. seniculus were determined by C.M.M. (Meireles, 1997). DNA extraction for A. caraya and A. seniculus was based on the protocol of Bell et al. (1981), whereas for A. belzebul and A. fusca, that of Sambrook et al. (1989) was used.

The g1 fragment was obtained using the PCR protocol described in Meireles et al. (1995), and consisted of an initial denaturation of 3 min at 94oC followed by 30 cycles of denaturation at 94oC (30 s), annealing at 55oC (45 s), extension at 72oC (45 s) and a final extension of 10 min at 72oC. The amplification products were separated by agarose gel electrophoresis. The DNA primers (synthesized at the Center for Molecular Medicine and Genetics, Wayne State University, Detroit, USA) were:

R1 5' -AAT GTG GAA GAT GCT GGG- 3' and

R2 5' -GTC ATG TCT GAG CAA CAA AC- 3'

Despite the presence of a 1.8-kbg1 deletion in all four species (Figure 1), these primers amplified a ~600-bp fragment in all cases.

Cloning of PCR products

The PCR products were separated in 1.0% agarose gels in TBE buffer (89 mM Tris-base, 89 mM boric acid and 2 mM EDTA, pH 8.0) containing 0.5 mg ethidium bromide/ml. The amplified fragments were excised and purified using a Qiaex II gel extraction kit (Qiagen). The purified DNA fragments were cloned into the pGEM-T vector system I (Promega) and transformed into Escherichia coli host JM109 (Promega) according to the protocol supplied by the vendors. Single-stranded DNA was prepared from selected clones using helper phage M13K07 (Promega) and purified by PEG-NaCl precipitation followed by phenol-chloroform extraction and ethanol precipitation.

Sequencing

Nucleotide sequences were determined for at least three clones of each species by the dideoxy chain-termination method (Sanger et al., 1977) using a Sequenase version 1.0 kit (United States Biochemical). The sequences were aligned by eye using version 3.0 of the ESEE sequence editor (Cabot and Beckenbach, 1989). Gaps were inserted to minimize the number of nucleotide substitutions and indels (insertions/deletions) needed to account for the descent of the aligned sequences. As PCR reactions commonly result in slightly different sequences for the same cloned fragments, a consensus sequence of three or more clones was defined for each species. These consensus sequences were then aligned against the known orthologous sequences which represented the outgroups for the phylogenetic analysis: Cebus albifrons (Cal), Macaca mulatta (Mmu) and Homo sapiens (Hsa).

Phylogenetic analysis

Phylogenetic analyses were performed using the maximum parsimony and maximum likelihood methods. The most parsimonious and the maximum likelihood trees were determined using the phylogenetic analysis using parsimony (PAUP) parsimony program, version 4.0 for DOS (written by David Swofford, Smithsonian Institute, Washington, DC, USA). For both trees, the strength of grouping values or Bremer decay indices were estimated using the PAUP program, and bootstrap analysis was applied to test the support for each grouping. Two thousand bootstrap replications with one shuffle per replication were carried out on the seven data sets. The number of parsimony-informative characters (synapomorphies) was established by eye and confirmed by the PAUP program. Divergence times were estimated using the branch lengths of the maximum likelihood tree constructed from a distance matrix calculated using Kimura's (1980) model. The molecular clock was calibrated using the local clock procedure, based on Goodman's (1996) estimate of 21 MYA for the divergence between Cebus and Alouatta.

RESULTS AND DISCUSSION

Analysis of g1-pseudogene sequences

PCR amplification using the 5' R1 and 3' R2 primers produced two fragments for all four genera. The fragment corresponding to g1 contained approximately 600 bp, and that of g2 about 2100 bp (Figure 2). The sequences indicated that this difference in size was due to a ~1.8-kb deletion at the g1 locus, which is shared by all four atelid genera (Meireles et al. 1995, 1998). These authors also encountered a ~150-bp insertion, described as a monomeric Alu element by Drs. Roy Britten (CIT, Pasadena, USA) and Jerzy Jurka (UC, Berkeley, USA), in A. caraya, A. belzebul and A. seniculus. In the present study, this same 150-bp Alu insertion was also found in A. fusca, indicating that it is an important synapomorphic marker for the analysis of Alouatta phylogeny and evolution. Figure 2 also shows a clear difference in the size of the g 1-globin fragment in two representative atelid genera, Ateles (~450 bp) and Alouatta (~600 bp).

The nucleotide sequences bordered by primers R1 and R2 in the seven primate species studied here spanned a total of 2,482 aligned base positions (Figure 3) consisting of the following g1 gene regions: the 5' coding region of exon 1 (1-35), intron 1 (36-160), exon 2 (161-211), a ~1.8-kb deletion (212-2092), a region between the deletion and insertion (2093-2149), a ~150-bp insertion (2150-2298), and the 3' flanking region (2299-2482). In the phylogenetic analysis, all sequences except that of A. fusca were also represented by a 1.8-kb region extending upstream from the R1 primer. The sequences in this region are described in Meireles et al. (1999). The DNA sequences were deposited in GenBank (Table I), and are also available from the authors on request, or can be accessed via internet at http://ns.med.wayne.edu/.

Phylogeny of Brazilian howler monkeys

The maximum parsimony consensus tree (Figure 4), constructed using the PAUP program, had a nucleotide substitution score of 813 (length). The numbers above the lines show the minimum number of additional substitutions required to break up the clade (strength of grouping - SOG - value or Bremer decay index), while the numbers below the lines show the bootstrap values (number of times the grouping was encountered in 2,000 replicates) as percentages. The arrangement of howler species was: {A. caraya [A. seniculus (A. fusca, A. belzebul)]}, with SOG values of 66, 2 and 2, respectively.

Figure 4 - Maximum parsimony tree derived from the aligned g1-globin nucleotide sequences in seven simian species. The length, or nucleotide substitution score, is 813. Values above the lines represent the minimum number of additional substitutions required to break up the clade designated by that node and those below the lines, the percentage of bootstrap values obtained in 2,000 replicates.

All bootstrap values were greater than the critical value of 75% proposed by Hillis and Bull (1993), with a minimum value of 82% for the A. seniculus-A. belzebul/A. fusca clade. The A. belzebul-A. fusca clade was supported by a value of 88%, while 100% of the replications identified A. caraya as the basal species. The number of shared-derived sequence changes (synapomorphies) found by the PAUP analysis also supported this arrangement. The positions of the synapomorphies for the Alouatta species clades (Figure 3) were:

The natural logarithm (ln) of the likelihood for the best maximum likelihood tree using the aligned g-globin sequences was -9329.13944. Bootstrap values (as a percentage of 2,000 replicates) are shown below the lines (Figure 5). Although the topologies of the tree and bootstrap values for the first (88) and last (100) clades were the same, the A. seniculus-A. belzebul/A. fusca clade had a borderline bootstrap value of 72. This analysis confirmed the cladistic groupings shown by the maximum parsimony tree (Figure 4).

Figure 5 - Maximum likelihood tree derived from the aligned g1-globin nucleotide sequences in seven simian species. Log likelihood is -9329.13944. Values above the lines represent the minimum decrease in the log likelihood required to break up the clade designated by that node and those below the lines, the percentage of bootstrap values obtained in 2,000 replicates.

Although the A. belzebul-A. fusca clade was relatively well defined, with a high bootstrap value (88%) and four shared synapomorphies, the analysis did not permit identification of the species closest to A. seniculus, despite the relative geographic proximity of A. belzebul in southwestern Amazonia (Hirsch et al., 1991). The data, nevertheless, supported the presence of a well-defined A. seniculus group [A. seniculus (A. belzebul, A. fusca)] as proposed by Hershkovitz (1949) and still widely accepted (e.g. Mittermeier et al., 1988). There is little doubt from these data that A. caraya should be placed in a distinct, monotypic group, and that Alouatta is monophyletic, with 15 synapomorphies.

The divergence times between the different Alouatta lineages (Table II) were calculated by the local molecular clock method, based on Goodman's (1996) estimate of 21 MYA for the split between Alouatta and Cebus, using the maximum likelihood branch lengths for the sequences of the g1-globin pseudogene. This analysis indicated that the different Alouatta lineages diverged relatively recently in relation to the putative + 11 MYA split of the alouattine and ateline groups (Meireles et al., 1999).